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Mg2+抑制表达在爪蟾卵母细胞上P2X4受体介导的ATP-激活电流

彭芳, 张玉芹^*, 曾燕, 周艳玲

武汉科技大学医学院生理学教研室，武汉 430065

摘要

本文旨在研究Mg2+对表达在非洲爪蟾卵母细胞上P2X4受体介导的ATP-激活电流(ATP-activated current, IATP)的调制及机理。大鼠P2X4受体野生型及突变体型cDNA在体外转录成cRNA，通过显微注射技术将该cRNA注入非洲爪蟾卵母细胞中进行表达。应用全细胞双电极电压钳技术研究Mg2+对IATP的影响。结果显示，细胞外液加MgCl2对IATP的影响如下：(1) Mg2+在0.5~10 mmol/L范围内，浓度依赖性地抑制IATP，半效抑制浓度(IC50)为(1.24 ± 0.07) mmol/L，冲洗10 min后，IATP可恢复；(2) 1 mmol/L的Mg2+使IATP量-效曲线右下移，最大效应电流(Emax)降低了(42.0 ± 2.1)%，但半数有效浓度(EC50)不变；(3) 预加Mg2+ 80 s后再加ATP，Mg2+对IATP抑制效应可达最大；(4) Mg2+对IATP的抑制不受膜电位的影响；(5)以100 μmol/L ATP激活的电流为对照，将P2X4受体280位的天冬氨酸突变成不带电荷的谷氨酰胺后，相同浓度的ATP诱发的IATP幅值仅为对照值的(4.12 ± 0.15)%；将280位的天冬氨酸突变成带负电荷的谷氨酸，IATP幅值和对照值相比没有显著性差异；(6) 当P2X4受体280位的天冬氨酸缺失后，100 μmol/L ATP诱发的IATP的幅值几乎为对照值的两倍，且0.5~10 mmol/L的Mg2+对相应的IATP无明显影响。以上结果提示：Mg2+抑制P2X4受体介导的IATP是非竞争性的、可逆的、具有浓度依赖性、时间依赖性、无电压依赖性；Mg2+的这种抑制效应可能是通过作用于P2X4受体280位的天冬氨酸而实现的。

关键词： Mg2+; ATP-激活电流; 爪蟾卵母细胞; 双电极电压钳技术

分类号：R338

[Mg(2+) inhibits ATP-activated current mediated by rat P2X4 receptors expressed in Xenopus oocytes.] [Article in Chinese]

PENG Fang, ZHANG Yu-Qin^*, ZENG Yan, ZHOU Yan-Ling

Department of Physiology, Medical College, Wuhan University of Science and Technology, Wuhan 430065, China

Abstract

To investigate the modulation of Mg2+ on rat P2X4 receptors and its underlying mechanism, we transcribed cDNA coding for wild-type and mutant P2X4 receptors to cRNA in vitro, injected the cRNA to oocytes of Xenopus laevis using the microinjection technique and revealed the effect of Mg2+ on ATP-activated currents (IATP) mediated by P2X4 receptors using the two-electrode whole-cell voltage clamp technique. The effects of extracellular Mg2+ on IATP were as follows: (1) In oocytes expressing P2X4 receptors, Mg2+ with concentration ranging from 0.5–10 mmol/L inhibited the amplitude of IATP in a concentration-dependent and reversible manner, with a 50% inhibitory concentration value (IC50) of (1.24 ± 0.07) mmol/L for current activated by 100 μmol/L ATP. (2) Mg2+ (1 mmol/L) shifted the dose-response curve for IATP right-downward without changing the EC50, but reduced the maximal current (Emax) by (42.0 ± 2.1)%. (3) After being preincubated with Mg2+ for 80 s, the inhibitory effect of the Mg2+ on IATP reached the maximum. (4) The inhibition of Mg2+ on IATP was independent of membrane potential from −120 mV to +60 mV. (5) Compared with the current activated by 100 μmol/L ATP in the wild-type P2X4 receptors, mutant P2X4 D280Q responded to the application of 100 μmol/L ATP with a smaller current. The peak current was only (4.12 ± 0.15)% of that seen in wild-type receptors. Mutant P2X4 D280E responded to ATP stimulation with a current similar to that observed in cells expressing wild-type receptors. (6) When Asp280 was removed from P2X4, the current amplitude of IATP was increased almost one-fold, and Mg2+ with concentration ranging from 0.5–10 mmol/L did not affect the IATP significantly. The results suggest that Mg2+ inhibits IATP mediated by P2X4 receptors non-competitively, reversibly, concentration-dependently, time-dependently and voltage-independently. The inhibitory effect of Mg2+ might be realized by acting on the site Asp280 of the P2X4 receptors.

Key words: Mg2+; ATP-activated current; Xenopus oocytes; two-electrode voltage clamp technique

收稿日期：2011-07-06　　录用日期：2011-11-03

通讯作者：张玉芹　　E-mail: zhangyuqin@wust.edu.cn

引用本文：

彭芳, 张玉芹, 曾燕, 周艳玲. Mg2+抑制表达在爪蟾卵母细胞上P2X4受体介导的ATP-激活电流[J]. 生理学报 2012; 64 (1): 75-81.

PENG Fang, ZHANG Yu-Qin, ZENG Yan, ZHOU Yan-Ling. [Mg(2+) inhibits ATP-activated current mediated by rat P2X4 receptors expressed in Xenopus oocytes.] [Article in Chinese]. Acta Physiol Sin 2012; 64 (1): 75-81 (in Chinese with English abstract).